Optimization of a β-sheet-cap for long loop closure.

Protein loops make up a large portion of the secondary structure in nature. But very little is known concerning loop closure dynamics and the effects of loop composition on fold stability. We have designed a small system with stable β-sheet structures, including features that allow us to probe these questions. Using paired Trp residues that form aromatic clusters on folding, we are able to stabilize two β-strands connected by varying loop lengths and composition (an example sequence: RWITVTI – loop – KKIRVWE). Using NMR and CD, both fold stability and folding dynamics can be investigated for these systems. With the 16 residue loop peptide (sequence: RWITVTI-(GGGGKK)2GGGG-KKIRVWE) remaining folded (ΔGU = 1.6 kJ/mol at 295K). To increase stability and extend the series to longer loops, we added an additional Trp/Trp pair in the loop flanking position. With this addition to the strands, the 16 residue loop (sequence: RWITVRIW-(GGGGKK)2GGGG-WKTIRVWE) supports a remarkably stable β-sheet (ΔGU = 6.3 kJ/mol at 295K, Tm = ~55 °C). Given the abundance of loops in binding motifs and between secondary structures, these constructs can be powerful tools for peptide chemists to study loop effects; with the Trp/Trp pair providing spectroscopic probes for assessing both stability and dynamics by NMR.

在自然界中，蛋白质环在二级结构中占很大比例。但是，关于环闭合动力学以及环组成对折叠稳定性的影响，人们知之甚少。我们设计了一个具有稳定β -折叠结构的小型系统，该系统具备能让我们探究这些问题的特性。利用在折叠时形成芳香簇的成对色氨酸残基，我们能够稳定由不同长度和组成的环连接的两条β -链（一个示例序列：RWITVTI - 环 - KKIRVWE）。利用核磁共振（NMR）和圆二色性（CD），可以对这些系统的折叠稳定性和折叠动力学进行研究。16个残基的环肽（序列：RWITVTI - (GGGGKK)₂GGGG - KKIRVWE）在295K时保持折叠状态（ΔGU = 1.6 kJ/mol）。为了提高稳定性并将系列扩展到更长的环，我们在环的侧翼位置添加了另一对色氨酸。通过在链上添加这一对，16个残基的环（序列：RWITVRIW - (GGGGKK)₂GGGG - WKTIRVWE）形成了非常稳定的β -折叠（在295K时，ΔGU = 6.3 kJ/mol，Tm = ~55°C）。鉴于结合基序以及二级结构之间存在大量的环，这些构建体对于肽化学家研究环的影响来说是有力的工具；色氨酸对为通过核磁共振评估稳定性和动力学提供了光谱探针。